Regulation of the transition from membrane repair attempts to lysophagy by SPG20/Spartin during the cellular response to lysosomal damage

Lysosomes are the main degradative organelles in the cell and serve as platforms for important signaling pathways. The loss of lysosomal function due to lysosomal membrane permeabilization (LMP) can be highly deleterious for the cell. LMP can be caused by different conditions such as lipid peroxidation, lysosomotropic drugs or disease-associated changes in lipid composition. Cells respond to LMP by lysosomal repair or if the damage is too extensive, selective macroautophagy of damaged lysosomes, termed lysophagy. However, at the start of this project it was not clear how the decision between repair and lysophagy of damaged lysosomes is made. In this project, the Troyer syndrome protein SPG20 (Spartin) was investigated as a possible regulator of transition from repair to lysophagy in human cells. SPG20 is a known interactor of the repair protein IST1 and a regulator of the Nedd4-like family of ubiquitin ligases. We found that SPG20 is recruited acutely to damaged lysosomes. Importantly, the recruitment dynamics of SPG20 and other factors involved in lysosomal damage response revealed that SPG20 was recruited shortly after the repair protein IST1 with increasing damage, but before the disintegration of lysosomes that exposes luminal glycans. During recovery following lysosomal damage, we detected SPG20 on individual lysosomes which were neither repaired nor re-acidified, and subsequently acquired the autophagy receptor Optineurin indicating the initiation of lysophagy. Consistent with this, the recruitment of SPG20 correlated with the emergence of lysine 63-linked (K63) ubiquitin chains on damaged lysosomes. We found that SPG20 on damaged lysosomes subsequently recruits and activates the ubiquitin ligase ITCH which mediates the K63-linked ubiquitylation of damaged lysosomes. In line with this, SPG20 and ITCH were found to be important for the K63-linked ubiquitylation of damaged lysosomes and the recruitment of the autophagy machinery for lysophagy initiation. An important element in SPG20-mediated initiation of lysophagy is the detection of damage on lysosomes by SPG20. We found that SPG20 binds IST1 via its MIT domain and senses lipid-packing defects in lysosomal membrane via its SC domain which is important for the recruitment of SPG20 to damaged lysosomes. The SC domain of SPG20 is predicted to contain four amphipathic helices which harbor bulky hydrophobic amino acids. These residues are typical of amphipathic lipid-packing sensors (ALPS) and aid in lipid-packing defect detection by ALPS. We found that lipid-packing defects occur on damaged lysosomes and SPG20 detects the lysosomal damage-associated lipid-packing defects via the SC domain. The bulky hydrophobic residues in the SC domain of SPG20 were found to be important for lipid-packing defect sensing by SPG20 and the subsequent initiation of lysophagy. In conclusion, the collected data suggests that SPG20 detects lipid-packing defects on damaged lysosomes and integrates this with IST1 binding in a possible coincidence detection mechanism. Thereby, SPG20 plays a prominent role in triaging of damaged lysosomes for lysophagy via ITCH-mediated ubiquitylation when the damage is extensive. In view of the link of SPG20 to the neurological disorder Troyer syndrome, our findings on how cells regulate the transition from lysosomal repair to lysophagy are particularly important for understanding the role of lysosomal function and damage response in pathophysiological conditions.